Project Summary: This work is proposed in response to NOT-AR-23-006 as a mechanistic study of innate and adaptive immune responses in systemic lupus erythematosus (SLE). SLE is a systemic rheumatic disease that disproportionately impacts females and racially and ethnically disenfranchised groups. Despite advances in treatment, the molecular mechanisms of SLE pathogenesis remain poorly understood. Cyclic G/AMP (cGAMP) synthetase (cGAS) is a central driver of the cell- intrinsic interferon (IFN) response which has been linked to SLE pathogenesis. When activated by cytosolic double-stranded DNA (dsDNA), cGAS generates cGAMP, a key second messenger for downstream IFN signaling. Resting cGAS is a monomer, and dsDNA-dependent activation entails dimerization and subsequent higher-order oligomer formation, resulting in the generation of phase-separated cGAS. In preliminary studies, we have identified the presence of autoantibodies targeting cGAS in SLE patient sera, which are associated with SLE disease activity. In addition, we have observed that cGAS can bind to and forms condensates on various nucleic acids, but dsDNA is the only ligand that permits the dimerization required for enzymatic activity and formation of hyrdo-gel-like condensates. Building upon these novel observations, we propose to test the hypothesis that biophysical changes triggered by cGAS activation shape the adaptive immune response that leads to the targeting of cGAS as an autoantigen in a subset of SLE patients with active disease. In Aim 1, we will test the theory that dsDNA-dependent gel-like cGAS condensates are preferentially targeted by SLE autoantibodies. To do this, we will develop assays incorporating purified cGAS (monomer vs dimer) complexed with various nucleic acid ligands (dsDNA, dsRNA, ssDNA, ssRNA, DNA-RNA hybrid). In Aim 2, we will determine if phase separation and/or ligand binding alter cGAS uptake and presentation by antigen presenting cells (APCs). To accomplish this, we will utilize monocyte-derived dendritic cells (moDCs) exposed to the same panel of cGAS-ligand complexes used in Aim 1, and will analyze cGAS uptake and subsequent moDC activation using confocal microscopy and flow cytometry. Then, we will examine the effect of nucleic acid binding on cGAS peptide presentation in the context of MHC II using a natural antigen processing assay (NAPA). We anticipate that this work will reveal interplay between innate and adaptive immune components that drives aberrant immune phenomena observed in SLE, providing a conceptual and experimental framework that could be readily applied to the numerous nucleic acid-binding autoantigens featured in SLE.